All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Propane, a major component of autogas or liquefied petroleum gas (LPG), is an emerging fuel for future energy supply and transportation. Propane is the third most widely used motor fuel and about 20 million tonnes of propane gas are used per year to fuel motor vehicles. It is estimated that propane provides heat and energy for more than fourteen million homes worldwide annually. Propane also has an existing global market for a wide number of other stationary and mobile applications, such as low emission vehicles, gas burners and refrigeration systems. Easy separation from liquid biotechnological processes as a gas and less energy requirements for liquefaction and storage, offers potential advantages to propane over other gaseous fuels.
Natural metabolic pathways for the renewable biosynthesis of propane do not exist. The discovery of an aldehyde deformylating oxygenase (ADO) from cyanobacteria, however, has paved the way for synthetic alkane pathways to be constructed [Schirmer A et al. Science 2010, 329:559-562; Akhtar M K et al. Proc Natl Acad Sci USA 2013, 110:87-92; Howard T P et al. Proc Natl Acad Sci USA 2013, 110:7636-7641]. A microbial platform for propane generation dependent on fatty acid biosynthesis was recently reported [Kallio P et al., Nature communications 2014, 5:4731]. Kallio P et al. concluded that the pathway was limited by total flux through fatty acid synthesis (FAS). The most obvious example of this limitation comes from the markedly enhanced rate of propane synthesis observed when fatty acids were supplied to the external media. Herein, the inventors sought to bypass this limitation by generating new synthetic pathways that are not dependent on FAS. The inventors designed a series of modified butyraldehyde pathways based on the CoA-dependent butanol pathways commonly found in Clostridium spp. Propane biosynthesis was thereafter achieved by interrupting the route to alcohol by the addition of ADO (FIG. 1).
The butanol pathway in Clostridium proceeds either via a keto acid route (Ehrlich pathway) or a CoA-dependent route. Higher yields of branched chain alcohols and aldehyde precursors (e.g. isobutyraldehyde) from the decarboxylation of keto acids make the Ehrlich pathway less attractive because ADO has a strong preference for straight chain aldehyde substrates. By contrast, butanol production by the CoA-dependent route initiates with the condensation of two molecules of acetyl CoA. Reduction in subsequent steps produces the end-product 1-butanol, via a butyraldehyde intermediate. There are several reports of engineered CoA-dependent butanol pathways in E. coli and other host organisms [Atsumi S et al. Nature biotechnology 2009, 27:1177-1180; Bond-Watts B B et al. Nat Chem Biol 2011, 7:222-227; Dellomonaco C et al. Nature 2011, 476:355-U131; Lan E I et al. J C Metab Eng 2011, 13:353-363; Lan E I. Proc Natl Acad Sci USA 2012, 109:6018-6023; Lan E I et al. Energ Environ Sci 2013, 6:2672-2681; Pasztor A et al. Biotechnology and bioengineering 2014]. Herein, the inventors constructed and evaluated a series of CoA-dependent butyraldehyde pathways that eliminate the dependency on AdhE2, thereby allowing butyraldehyde to be re-routed towards propane instead of butanol.
The use of ADO (from Prochlorococcus marinus MIT9313) as a terminal decarbonylase has been used for the production of medium/long chain (C9-C17) as well as short chain-length alkanes (C3, C7). Variant forms of ADO have demonstrated improved activity with the shorter chain aldehydes that are not encountered in native cyanobacteria. These variant forms of ADO are therefore attractive enzyme components for building new synthetic pathways with a greater productivity, as addressed herein.
Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways derived from FAS recently engineered in E. coli. 